Tautomer-Selective\nFluorescence Spectroscopy of Oxyluciferin\nAnions

Bioluminescence in fireflies and related insects arises\nas emission\nfrom the fluorophore oxyluciferin, yet the color of the emission in\nthese insects can range from red to green. The chromophore’s\nmicroenvironment or multiple tautomeric forms may be responsible for\nthe color tuning; however, these effects are difficult to separate\nin condensed phases. To investigate the role of oxyluciferin tautomerization\nin the color tuning mechanism, gas-phase spectroscopy eliminates solvent\neffects and allows us to study the fluorescence from individual tautomers.\nUsing a home-built mass-spectrometry setup with a cylindrical ion\ntrap cooled with liquid nitrogen, we measure fluorescence from the\nenol-locked form of oxyluciferin in the gas phase and characterize\nthe photophysics of both keto and enol forms. At 100 K, the enol-locked\nform has an emission maximum of 564 ± 1 nm, coinciding with a\npreviously reported assignment in oxyluciferin. We measure the absorption\nspectrum and find a maximum at 560.5 ± 0.5 nm, which implies\na Stokes shift of 110 cm<sup>–1</sup>. The absorption spectrum\nis compared to Franck–Condon simulated spectra that identify\none dominant vibrational mode in the transition. Additionally, we\nultimately separated the emission by the enol and keto forms present\nin the trap by selectively exciting each form. We demonstrate that\nfluorescence measured close to the 0–0 transition limits the\nreheating of the ions, thereby providing the coldest ions and therefore\nthe narrowest emission spectra. These experimental data are also crucial\nbenchmarks for computational studies, offering actual emission spectra\nin the gas phase for both tautomeric forms. Thus, our findings serve\nas essential reference points for excited-state calculations aimed\nat understanding the color tuning mechanism of bioluminescence computationally.